title: Effect of ionic strength and pH on the physical and chemical stability of a monoclonal antibody antigen-binding fragment.
abstract: Monoclonal antibody (mAb) fragments are emerging as promising alternatives to full-length mAbs as protein therapeutic candidates. Antigen-binding fragments (Fabs) are the most advanced with three Fab-based drug products currently approved. This work presents preformulation characterization data on the effect of pH, NaCl concentration, and various cationic excipients on the physical and chemical stability of a Fab molecule with multiple negatively charged Asp residues in the complementarity-determining region. Conformational stability was evaluated using an empirical phase diagram approach based on circular dichroism, intrinsic Trp and extrinsic 8-anilino-1-naphthalene sulfonate (ANS) fluorescence, and static light scattering measurements. The effect of NaCl concentration, various cationic excipients and pH on the Fab molecule's conformational stability, aggregation propensity, and chemical stability (Asp isomerization) was determined by differential scanning calorimetry, optical density measurements at 350 nm (OD350 ), and ion-exchange chromatography, respectively. Increasing NaCl concentration increased the overall conformational stability, decreased aggregation rates, and lowered the rates of Asp isomerization. No such trends were noted for pH or cationic excipients. The potential interrelationships between protein conformational and chemical stability are discussed in the context of designing stable protein formulations.
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas 66047, USA.
authors.forename: Tingting
authors.initials: T
authors.lastname: Wang
authors.forename: Ozan S
authors.initials: OS
authors.lastname: Kumru
authors.forename: Li
authors.initials: L
authors.lastname: Yi
authors.forename: Y John
authors.initials: YJ
authors.lastname: Wang
authors.forename: Jennifer
authors.initials: J
authors.lastname: Zhang
authors.forename: Jae Hyun
authors.initials: JH
authors.lastname: Kim
authors.forename: Sangeeta B
authors.initials: SB
authors.lastname: Joshi
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
authors.forename: David B
authors.initials: DB
authors.lastname: Volkin
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry*; D002412:Cations / Q000737:chemistry; D005079:Excipients / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D009994:Osmolar Concentration; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D002412:Cations; D005079:Excipients; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D012965:Sodium Chloride
keywords: Asp isomerization; IgG antibodies; antigen-binding fragments (Fab); chemical stability; formulation; monoclonal antibodies; physical stability; protein aggregation; salt; stabilization